Information about the structure and replication of human immunodeficiency virus type 1 (HIV-1) has provided a rationale for the development of a number of antiviral drugs. It is prudent to continue exploring regulation of all phases of the virus life cycle in order to develop new approaches for inhibiting replication of drug-resistant HIV-1 variants. To this end, the regulation of HIV-1 alternative splicing, a step in the virus life cycle that is only beginning to be understood will be studied. Viral mRNAs are present at very different abundances in the HIV-1 infected cell which implies that splice site utilization is regulated. Cis elements and cellular factors regulating splicing of tat, vpr, rev and env/nef mRNAs will be studied. Several cis elements (exon splicing silencers) within tat exon 2 and 3 have been characterized that act to inhibit splicing at the tat 3'splice sites. In vitro splicing assays will be used to identify cellular factors that bind to these elements and mediate the inhibition. The effect of mutations in these elements will be studies in the context of number prototype group M HIV-1 strains in which the tat exon splicing silencers are conserved and in a prototype group O or outlier strain lacking these elements. RNA spliced at the vpr 3' splice site is also present at low abundance in infected cells. In vitro splicing assays and in vivo transfection analysis will be used to identify elements inhibiting splicing at this splice site. The most abundant viral mRNAs are spliced at the rev and env/nef 3' splice sites. It has been shown that one of the rev splice sites overlaps an env/nef splicing branchpoint. Thus, splicing factors may compete for the same site on the viral RNA and this may regulate the balance between rev and env/nef mRNA splicing. This model will be tested and the competing cellular factors will be identified. Studies on the splicing regulation of HIV-1 and the role of cellular factors should contribute to our understanding of viral replication in diverse cell types.